CHAPTER ONE: INTRODUCTION 1.1 IMMUNE SYSTEM The immune system is an intricate defense mechanism comprised of biological elements that protect the organism against foreign invaders (like bacteria, viruses, etc.) by recognizing and destroying them. The immune system is classified based on the time and nature of their actions; the first section of the immune system is composed of a layer of epithelial cells that acts as a physical barrier by providing an immediate defense against the invading pathogens in a non-specific manner and is termed as innate immune system. Innate immunity is triggered by the incoming pathogens that are identified by the pattern recognition receptor (PPR). The PPR identifies the incoming pathogen by recognizing a pattern specific for each group of microorganisms termed as pathogen-associated molecular patterns (PAMPs) invading the host [1]. After recognizing the pathogens, these epithelial cells secrete chemicals (enzymes like lysozyme) to carry out cell wall lysing of the pathogen. The cell lysing is then followed by engulfing and destroying the pathogen by endocytosis and phagocytosis. The second component of the immune system is called the adaptive immune system. Adaptive immunity is more specific to the invading pathogens. This type of immunity is characterized by immunological memory, through which each pathogen is recalled. What helped to eradicate the infectious pathogens is also recalled from the lymphocytes [2] . The lymphocytes belong to the category of leukocytes that are mainly concentrated in the central lymphoid systems such as the spleen and lymph nodes. 1 The two components of the immune systems complement each other very effectively. In summary, the non-specific innate immunity is developed immediately after birth and is effective only when challenged whereas the specific adaptive immunity is developed by an organism over a period of time by exposing it to immunization and is effective for a long time period as it utilizes immunological memory. 1.2 CELLS INVOLVED IN IMMUNE RESPONSE The inflammatory reaction is the result of immune response. It is stimulated when the entering pathogen injures the host tissue, which in turn recruits cells by secreting chemokines. Some of the essential components that are responsible for the pro-inflammatory response are TNF-α, cytokines like IFN- , white blood cells like antigen presenting dendritic cells, lymphocytes like T cells and B cells, and effector cells or natural killer cells (NK cells) [3]. Figure 1 gives a clear diversification of the blood stem cell into various immune cells that carry out the immune response effectively. Out of these components, T cells play a significant role in immune response. These are divided into two major classes: T helper cells (CD4+ cells) and cytotoxic T cells (CD8+ cells) [4]. 2 Figure1: Cells involved in the immune response (Terese Winslow, 2007). Out of these cells, the cells that encompassed in the lymphoblast lineage mostly involved in immune response. 1.3 T-HELPER CELL DEVELOPMENT AND ACTIVATION The T helper cells are a subset of lymphocytes that act against the pathogens by stimulating cytokines (especially IFN-), leading to an inflammatory response. This category of T cells has CD4+ proteins on their surface. The activation of this cell lineage is regulated by the antigen peptides binding to the major histocompatibility complex class II (MHC-II) present on the surface of antigen presenting cells (APCs) [5]. Additionally, these T helper cells mature and develop in the thymus region [6]. The T helper cells also differentiate into many sublineages, like Th1, Th2, Treg, Th17, T follicular helper (Tfh) cells, and Th9 (IL-9-expressing) cells. Of these sublineages Th1 and Th2 have a more 3 established participation in the immune response mechanism (Figure 2) [7]. Figure2: T-helper cell lineages [8] . The activated CD4+ T cell differentiates into many T-helper cell lineages which are mediated by different interleukin molecule (Example: The Th1 lineage is mediated by IL-12 whereas Th2 lineage is mediated by IL-4). The cytokine IFN-γ stimulates the macrophages and dendritic cells to produce interleukin-12, which is critical in promoting the Th1 cell lineage development. The IFN-γ cytokine stimulation also inhibits the production of other interleukins (like IL-4, IL-10), thus preventing the development of other Th cell lineages such as Th2, Treg, etc. 4 1.3.1 TRANSCRIPTION REGULATION IN Th1 CELL DEVELOPMENT Transcription regulation is a dynamic process that regulates the expression of genes by employing various transcription regulatory elements such as activators, enhancers, and repressors. One of the complex transcription regulatory networks in higher eukaryotes is the immune cell transcription regulatory network. The T cell transcription regulatory network is a complex system regulated by various families of factors, such as NF-B, STAT family, and GATA (FIGURE 3) [9]. The transcription regulatory network plays an essential role in diversification of the T cell lineage into Th1 and Th2 sublineages. The Th1 lineage‟s transcription network is regulated by T-bet which cooperates with another factor called STAT4 [10]. Figure3: Transcription regulation in Th1 cell [11].The T-bet induces IL-12 expression that enables the STAT4 to undergo dimerization process leading the Th1 cell development. 5 T-bet (also called Tbx-21) is a member of the T-box family of transcription factors (TFs). It plays an essential role in the activation of IFN-γ production, which eventually leads to the repression of interleukin-4 and 5 (IL-4 and IL-5); this plays a major role in the development of Th 2 cell lineage [12]. T-bet also induces the expression of IL-12R, enabling STAT4 activation for Th1 cell development [13]. STAT4 is a member of the STAT transcription factor family that is regulated by a cytokine response [14]. The TCR signal transduction cascade activates the master regulators and also the cytokine gene framework to induce the transcription process leading to the development of Th1 cell lineage [15]. 1.4 ROLE OF CIS-REGULATORY MODULE IN TRN (TRANSCRIPTION REGULATORY NETWORK) A few transcription factors (for example, T-bet and STAT4) work in coordination with each other to regulate the transcription regulatory network. Such factors bind to the specific transcription factor binding sites (TFBS) that are located very close to each other in the genomic region. These co- occurring heterotypic or homotypic clusters of TFBS form the base for the transcription regulatory network and are referred to as Cis-regulatory modules (CRM) [16]. The CRMs are the main components that control the regulation of transcription regulatory networks [17]. The CRM is about 500-1000 bp in length and is mostly positioned in the upstream region to the transcription start site. 6 Figure 4: Representation of the cis-regulatory module or Cistrome unit. In this representation, the transcriptions factors Sp1, c-Myc/Max and others binding to their respective TFBS on the genes forming the cis-Regulatory Module or unit. And these sites co-regulated to carry out gene expression. 7 CHAPTER TWO: BACKGROUND 2.1 STAT4 BIOLOGY STAT4 transcription factors are categorized under the family of signal transducers and activators of transcription proteins that play a cardinal role in the development of Th1 cell lineage. The activation of STAT4 molecules is an orchestra of interactions with other molecules. The IL-12 molecule binds to the IL-12 receptor on the surface of the naive T helper cell (Th0); this binding in turn mediates the conversion of the inactive STAT4 molecule to an active homodimer STAT4 molecule. The active STAT4 molecule carries the signal from the cell surface to the nucleus, and it then binds to various cis-regulatory modules that control the transcription of a number of genes involved in Th1 cell development. STAT4 has many similar functional domains that belong to the STAT family of proteins, such as the DNA-binding domain, a conserved SH2 domain that is involved in dimerization processes and a C- terminal transactivation domain [18]. 8 Figure 5: Biology of STAT4 involvement in Th1 cell development. The activation of STAT4 is mediated by the Jak-STAT (Janus kinase-signal transducers and activators of transcription pathway). In higher eukaryotes, the Jak-STAT pathway initiates the signaling cascades for the stimulation of many cytokines and growth factors. The conversion of homodimer of STAT4 molecules to a heterodimer molecules is also advocated through the Jak- STAT by enhancing the phosphorylation process, which in turn stimulates the conversion of naive Th cells (Th0) to a mature and active state [19]. 9 Figure 6: JAK-STAT (Janus kinase/signal transducers and activators of transcription) pathway. The highlighted circle depicts how the STAT dimerization is carried out in the Jak-STAT pathway. (Courtesy: KEGG Database) 10 2.2 CURRENT UNDERSTANDING OF STAT4 Numerous wet lab biological studies have been conducted to decipher the transcription regulation process of the immune system. For example, one study demonstrates the early target genes of IL-12 and STAT4 signaling in Th1 cell differentiation [20], and another study deals with the requirements for the splice forms STAT4α and STAT4β to mediate responses to IL12 [21]. In addition to these in vivo studies, there was also some in vitro computational analysis performed to understand the gene transcriptional mechanism of the immune system. Some of the latest computational studies were performed in order to obtain an elaborated knowledge about the significance of STAT4 molecules in Th1 cell development, which will be discussed in this section. A significant study by Lai et al. deals with the role of STAT4 and STAT6 in epigenetic modification and transcriptional regulation in Th1 cell development. The study analyzes the STAT4 and STAT6 histone methylation ChIP-seq data to investigate the transcriptomics of murine STAT4-mediated Th1 development. The histone methylations focused on in this study were H3k4me3 and H3k27me3.